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Expression of nucleolar protein p120 predicts poor prognosis in patients with stage I lung adenocarcinoma
Annals of Oncology 12. 1121-1125. 2001. © 2001 Khmer Academic Publishers. Printed in the Netherlands.
Original article Expression of nucleolar protein pi20 predicts poor prognosis in patients with stage I lung adenocarcinoma Y. Saijo,1 G. Sato,1 K. Usui,1 M. Sato,2 M. Sagawa,2 T. Kondo, 2 Y. Minami 3 & T. Nukiwa1 ^Department of Respiratory Oncology and Molecular Medicine. 2 Department of Thoracic Surgery. Institute of Development. Aging, and Cancer. Tohoku University: ''Division of Epidemiology. Miyagi Cancer Center, Natori. Japan
Summary Background: P120 is a proliferation-associated nucleolar protein found in most human malignant tumors, but not in resting normal cells. In our previous studies, the expression of pl20 was statistically correlated with the proliferation capacity in human lung cancer cells and could be a prognostic marker for resected lung adenocarcinoma. Patients and methods: The expression levels of pl20 in tumors were assessed by immunohistochemistry in 59 patients with stage I lung adenocarcinoma who underwent radical resection. Using clinical follow-up data, the prognostic significance of pi20 calculated by labeling indices was evaluated using Cox's proportional hazard model. Results: A mean ± SD of the labeling index of pl20 was
Introduction Lung cancer is a major cause of cancer death worldwide. In Japan, deaths from lung cancer increase annually and accounted for more than 50,000 deaths in 1998 [1]. The overall five-year survival rates remain less than 20% [2]. Although the patients with stage I lung cancer show the best prognosis, about 30% relapse after surgical resection. Therefore, it would be important to identify stage I patients with an adverse prognosis and treat them with adjuvant chemotherapy. An additional predictor other than that of the TNM classification for identifying the patients with a high risk of recurrence in early stage non-small-cell lung cancer should be formulated to complement the current methods. PI 20, identified as a proliferation-associated nucleolar protein, is detected in a wide range of human tumors but not in quiescent cells or normal resting cells [3, 4]. Gene transfer experiments of pl20 into mammalian cells indicate a linkage between pl20 expression and the growth capacity of tumor cells [5, 6]. Nop2, a recently identified yeast homologue of human pl20, is required for the synthesis of the 60S ribosomal subunit and processing of the 27 S pre-rRNA to the mature 25 S rRNA [7-10]. Although no specific biochemical function of pl20 in eukaryotic cells has been assigned, the increased expression of p!20 in tumor cells suggests
35.3 ± 14.4%. No significant correlation was found between the expression levels of pi20 and clinicopathological factors. Using a cutoff value of 35% in the labeling index of pi20, patients with high expression of pl20 experienced early recurrence and shorter survival compared with those having low expression of pl20 (P = 0.04). Multivariate analysis revealed that pl20 served as an independent and strongest prognostic factor for resected lung adenocarcinoma (P = 0.033). Conclusion: This article provides the first evidence that the expression levels of pi20 in tumor tissues can be used as an independent and powerful prognostic marker for resected stage I lung adenocarcinoma. Key words: lung adenocarcinoma, nucleolar protein, pl20, prognosis, stage I
pl20 takes part in activated ribosomal biogenesis and accelerated cell growth. We have previously studied the significance of pl20 expression in human lung cancer in vitro and in vivo [11, 12]. The expression of pl20 was statistically correlated with the proliferation capacity in human lung cancer cells [11]. In 74 patients with lung adenocarcinoma, the expression levels of pi20 in tumor tissues evaluated by immunohistochemistry could predict the prognosis of patients with resected lung adenocarcinoma [12]. However, these patients comprised different stages (39 with pathologic stage I, 16 with pathologic stage II, and 19 with pathologic stage HI) and the median follow-up time was relatively short (38.5 months). Therefore, our purpose was to study the prognostic significance of pi 20 in lung adenocarcinoma at an early stage and with a longer follow-up in another set of patients with stage I lung adenocarcinoma. Our results demonstrate that the expression of pi20 is an independent prognostic marker in stage I lung adenocarcinoma. Patients and methods Patients and tissue samples We studied 59 patients with stage I lung adenocarcinoma who consecutively underwent surgical resection in the Hospital of the Institute of
1122 Development. Aging, and Cancer, Tohoku University, and affiliated hospitals from February 1991 to July 1996. All patients underwent surgery without pre-operative treatment. Informed consent was obtained from all patients before surgical resection. The patients consisted of 30 men and 29 women whose ages ranged from 45 to 76 years. According to the International System for Staging Lung Cancer [13], the cases comprised 42 patients with pathologic stage IA and 17 with pathologic stage IB. For the present analysis, the follow-up data were updated in July 2000 A minimum 48-month follow-up was obtained. During the follow-up. 15 patients died of lung cancer For routine histologic studies, tissues fixed with 10% formaldehyde were stained with hematoxylin and eosin.
lmmunohistochemical staining of pi 20
Fresh tumor samples were embedded in an ornithine carbamil transferase (OCT) compound (Tissue-Tek. Elkhart, Indiana), frozen in liquid nitrogen and stored at — 80 °C until use. Cryostat specimens were fixed with 2% formaldehyde and permeabilized with acetone. The samples were then incubated with mouse anti-human pl20 monoclonal antibody (pl20 MAb) (Oncogene Science, Cambridge, Massachusetts) overnight at 4°C [II]. lmmunohistochemical staining was performed using the streptavidin-biotin procedure with a Histofine SAB PO (M) kit (Nichirei, Tokyo). The tissues were finally counter-stained with Myer-hematoxylin solution Two examiners independently observed more than 500 cancer cells in more than 10 randomly selected fields and counted the pi 20 positive cancer cells. The labeling index of p!20 was defined as the percentage of cancer cells with pi 20 positive nucleoli among the total number of cancer cells counted. When a difference of the labeling index of pl20 was over 15% between two examiners, the index was re-evaluated. The results of the assessment for pl20 staining were blinded concerning the clinical data of the patients.
Statistical analysis All statistical analyses were carried out using the Abacus Concepts. Slat View (Abacus concepts. Inc.. Berkley, California). The chi-square test was used to examine the association between the labeling index of pl20 and several clinicopathological factors. The overall survival was calculated from the date of surgery, using as the end point death from any causes. Disease-free survival (DFS) was determined from the day of surgery, using as the end point the initial recurrence of the resected lung cancer assessed by clinical examinations. No patients died of other disease or were lost to follow-up. Overall survival and DFS curves were calculated using the Kaplan-Meier method and analyzed by the log rank test. The influence of each variable on survival was examined by the Cox's proportional hazards model in multivariate regression analyses. Statistical significance was defined as P < 0.05.
Results lmmunohistochemical staining of p 120 and clinicopathologicalfactors Nucleolar protein pl20 was clearly stained on the nucleoli of cancer cells by immunohistochemistry (Figure 1). In contrast, only nucleoli of alveolar macrophages were weakly stained in normal lung tissues. The expression levels of pl20 protein were quantified by counting the pl20-positive cancer cells in the tumor tissues. The labeling indices of pi20 among the tumor tissues varied from 9.7% to 64.7%, with a mean of 35.3 ± 14.4%. These indices were quite similar to those of our previous study
Figure I. lmmunohistochemical staining of nucleolar protein pl20 in resected lung adenocarcinoma. A frozen section of resected lung adenocarcinoma was stained with mouse monoclonal antibody pi20 as described in 'Patients and methods'. P120 was overexpressed in nucleoli of the cancer cells, but not in those of stromal (magnification x400).
[11]. No clinicopathological factors including gender, age, tumor size, and differentiation, were statistically correlated with the labeling index of pi 20 (Table 1). Expression ofpi 20 and prognosis in lung adenocarcinoma with stage I During the follow-up period (up to 60 months), 15 patients died of lung cancer. Post-operative overall survival and DFS curves in all cases according to the labeling indices of pl20 are shown in Figures 2a and 2b. When the cutoff value of the labeling index of pi 20 was chosen at 35%, patients with a high expression of pl20 showed a worse prognosis than those with low expression of pl20 (P - 0.04) (Figure 2a). Although median Table 1 Clinicopathological factors in 59 patients with stage I lung adenocarcinoma and labeling index of pi 20 in resected tumor tissues.
Gender Male Female Age < 65 5= 65 Tumor size Tl T2 Differentiation Well Moderate Poor Other
No. of patients
Labeling index ofpl20
29 30
36 9 ± 12.3" 33.9 ± 16.3
NS
28 31
36.4 ± 14.6 3 4 4 + 14.6
MS
42 17
36.2 ± 13.5 32 2 ± 16.7
34 12 9
35.4 ± 36.6 ± 31 5 ± 39 4 ±
4
Abbreviation' NS - not significant. ' Mean ± standard deviation.
15.4 12.7 13.6 86
P-value
NS
1123 Table 2. Mullivariate regression analyses of variables for predicting overall survival in patents with stage I lung adenocarcinoma.
(a) Lowpl20(n=28)
Variable
Hazard ratio
Unfavorable/favorable
f-value
Age Sex Tumor size Differentiation P120
2 607 0.158 2.958 0.675 10.417
>65/^65 male/female T2/T1 moderate, poor/well >35%/=S35%
0.183 0 111 0.111 0.412 0.033
Table 3 Multivariate regression analysis of variables for predicting disease free survival in patents with stage I lung adenocarcinoma. 10
20 30 40 Monthi after operation
50
(b)
Variable
Hazard ratio
Unfavorable/favorable
P-value
Age Sex Tumor size Differentiation P120
1.647 0.111 6.297 0.489 19.262
>65/<65 male/female T2/T1 moderate, poor/well > 35%/ s: 35%
0.498 0.074 0.024 0 484 0.012
Discussion
20 30 40 Monthi after operation
50
60
Figure 2. The Kaplan-Meier curves according to nucleolar protein pi 20 expression in patients with stage I lung adenocarcinoma Postoperative overall survival (A) and disease free survival (B) curves in 59 patients with stage-I lung adenocarcinoma were plotted based on a comparison between low (<35%) (28 cases) and high (3s 35%) (31 cases) labeling indices of pl20
survival times have not been reached in both the high pl20 expression group and the low pl20 expression group, the three- and five-year overall survival were 95% and 89% in the low pl20 expression group, but were only 77% and 65% in the high pl20 expression group. The patients with high pi20 expression tended to relapse earlier with high incidence resulting in shorter prognosis, but the p value of DFS was not significant (P = 0.07) (Figure 2b). The factors affecting the overall survival and DFS were analyzed by multivariate analysis using Cox's proportional hazards model. For overall survival, only pi20 was an independent prognostic factor (hazard ratio = 10.417, P = 0.033) among all variables (Table 2). Although both tumor size and pi20 were independent prognostic factors for DFS (Table 3), the hazard ratio of pl20 (19.26) was larger than that of the tumor status (6.297). Taken together, the expression of pi 20 was the strongest prognostic factor in the stage I lung adenocarcinoma.
The purpose of this study was to evaluate whether the expression level of pl20 in tumor tissues could predict the clinical outcome of patients with stage I lung adenocarcinoma. Our current study indicates that the expression of pi20 correlates with an early recurrence and poor prognosis after surgery. P120, a proliferation-associated nucleolar protein, is closely associated with the proliferative properties of mammalian cells. In human cancer cell lines, the expression of pl20 was documented to be associated with the S phase fraction of the cell cycle and doubling time [14]. The over-expression of pl20 in NIH3T3 cells by pl20 gene transfection enhanced cell proliferation [5]. In addition, down-regulation of pi20 expression by antisense pl20 gene caused growth inhibition of cancer cells [6, 15]. P120 was overexpressed more than 10-fold in tumor cells compared to normal resting cells. Although the specific transcriptional factor for pl20 has not been identified, the regulatory mechanisms of pi20 gene expression have been studied [16-18]. Mapping of the cisacting transcriptional control elements for the pl20 gene showed that, when the region containing the Alu sequences was deleted, transcription of the pl20 gene was reduced six-fold [16]. A recent report provides additional evidence that an RNA polymerase III transcriptional unit containing a homology to the highly repeated Alu sequence is located in the 5' flanking elements of the pl20 gene and that this positively regulates pi20 gene expression [18]. In situ expression of pi20 in malignant tissues has only been studied in a few human cancers including breast, prostate, colon, and lung cancer [11, 12, 19-23]. Freeman was the first investigator to study the expres-
1124 sion of pl20 in human cancer tissues and demonstrate a correlation between pl20 and the prognosis [19]. In 114 human breast cancer patients, they reported that 69% of the breast cancer tissues were positive for pi20, and that the immunopositivity of pl20 was correlated with patient survival, nodal status, estrogen receptor levels, and number of mitoses. Multivariate analysis showed that the worst prognosis was for patients who had tumor-positive nodes and tumor expressing pl20. This study concluded that pl20 was an independent and strongest prognostic marker in resected breast cancer. Ueki et al. reported increasing pl20 expression in accordance with the progression from mild dysplasia to severe dysplasia and carcinoma in human colorectal tumors [21]. They also demonstrated a significant correlation between pi 20 expression and another cell proliferation marker, Ki-67, but not with any known prognostic markers in colorectal carcinomas. Recently, the prognostic significance of pl20 in prostate carcinoma was reported [23]. One hundred thirty-two specimens of prostate carcinoma were stained with pl20 along with other proliferation markers. The expression of pl20 correlated with the post surgical recurrence in univariate analysis, but not in multivariate analysis. We have studied the expression of pl20 in human lung cancer using cancer cell lines and resected tumors. The expression of pi20 in six lung cancer cell lines was statistically correlated with their proliferation capacities [11]. In our previous immunohistochemical study of pi20 in resected lung adenocarcinoma, the expression level of pi20 was negatively correlated with the tumor doubling time (DT) determined by a retrospective radiographic calculation. Patients with a high labeling index for pi 20 had a high risk of early recurrence, resulting in a poor prognosis [12]. This study clearly shows that the expression of pl20 has prognostic value in stage I lung adenocarcinoma. The patients with a low labeling index of pl20 (<35%) had a statistically better survival rate than those with a high labeling index (^35%) (overall survival rate; 89% vs. 65%, P = 0.04). The multivariate analysis indicated that pi20 expression is the only prognostic indicator for overall survival and that pl20 expression and T status are significant indicators for disease-free survival in stage I lung adenocarcinoma. Many genetic markers have been analyzed as independent prognostic factors in stage I lung adenocarcinoma, such as K-ras, p53, microsattelite alterations, PCNA, Ki-67, etc. [23-26]. PCNA, ki-67 and pl20 are all proliferation markers. However, PCNA and ki-67 are localized in the nucleus whereas pl20 are localized in the nucleolus. Although prognostic value of pl20 in stage I lung adenocarcinoma was shown in this study, a further study in another set of patients should be evaluated to confirm the prognostic value of pl20. In addition, correlation of pl20 and other genetic markers should be studied in lung adenocarcinoma. Finally, we conclude that pl20 could predict the clinical outcome of the patients with stage I resected
lung adenocarcinoma by reflecting the tumor growth fractions.
References 1. Lung Cancer Registration Committee. Time trends and survival rate of lung cancer. In Watanabe S, Timonaga S, KakizoeT (eds): Gann Monograph on Cancer Research 1995, 43: 119-127. Tokyo: Japan Scientific Societies Press. 2 Freeman JW, Busch RK, Gyorkey F et al. Identification and characterization of a human proliferation-associated nucleolar antigen with a molecular weight of 120,000 expressed in early Gl phase. Cancer Res 1988; 48: 1244-51. 3. Busch H. The final common pathway of cancer: Presidential Address. Cancer Res 1990; 50: 4830-8. 4. Perlaky L, Valdez BC, Busch RK et al. Increased growth of NIH3T3 cells by transfection with human pl20 complementary DNA and inhibition by a pl20 antisense construct 1992; Cancer Res 52: 428-36. 5. Saijo Y, Perlaky L, Valdez BC et al The effect of antisense pl20 construct on pi 20 expression and cell proliferation in human breast cancer MCF-7 cells. Cancer Lett 1993; 68: 95-104. 6 Beus E, Brockenbrough JS, Hong B et al. Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker. J Cell Biol 1994; 127- 1799-813. 7. Hong BJ, Brockenbrough S,Wu Pet al Nop2p is required for prerRNA processing and 60S nbosome subunit synthesis in yeast. Mol Cell Biol 1997; 17: 378-88. 8. Gustafson WC, Taylor CW, Valdez BC et al. Nucleolar protein pl20 contains an arginine-nch domain that binds to ribosomal RNA. Biochem J 1990; 331: 387-93. 9. King M, Ton D, Redman KL. A conserved motif in the yeast nucleolar protein Nop2p contains an essential cysteine residue. Biochem J 1999; 337: 29-35. 10. Uchiyama B, Saijo Y, Kumano N et al. Expression of nucleolar protein pl20 in human lung cancer: Difference in histological types as a marker for proliferation Clin Cancer Res 1997; 3: 1873-7. 11 Sato J, Saijo Y, Uchiyama B et al. Prognostic value of nucleolar protein pl20 in patients with resected lung adenocarcinoma. J Clin Oncol 1999; 17: 2721-7. 12. U1CC In Sobin LH, Wittekind Ch (eds): TNM Classification of Malignant Tumours, fifth edition New York: Wiley-Liss 1997. 13. Fonagy A, Swidersk C, Ostrovsky AM et al Effect of nucleolar pl20 expression level on the proliferation capacity of breast cancer cells. Cancer Res 1994; 54: 1859-64. 14. Valdez BC, Perlaky BC, Saijo Yet al A region of antisense RNA from human pl20 cDNA with high homology to mouse pl20 cDNA inhibits N1H3T3 proliferation. Cancer Res 1992; 52: 5681-7. 15. Haidar MA, Henning D, Busch H. The upstream sequence -537 to -278 is necessary for transcription of the human nucleolar antigen pl20 gene. Mol Cell Biol 1990, 10. 3253-5. 16. Haidar MA, Henning D, Busch H. Spl is essential and its position is important for pl20 gene transcription- A 35 bp juxtaposed positive regulatory element enhances transcription 2.5 fold. Nucleic Acids Res 1991; 19: 6559-63. 17. Lowe D, Henning D, Reddy R. An RNA polymerase III transcription unit located in the upstream control regions of the human proliferating-cell nucleolar protein pi 20 gene is transcribed m vitro and in vivo. Eur J Biochem 1999; 266- 112-22. 18. Freeman JW, McGrath P, Bondada V et al. Prognostic significance of proliferation associated nucleolar antigen pl20 in human breast carcinoma. Cancer Res 1991; 51: 1973-8. 19. McGrath PC, Holley DT, Hamby LS et al Proliferation-associated nucleolar antigen pl20' A prognostic marker in node-negative breast cancer. Surgery 1994; 116' 616-21. 20. Ueki T, Nakayama Y, Sugao Y et al. Significance of the expression
1125
21.
22.
23.
24.
25.
of proliferation-associated nucleolar antigen pi 20 in human colorectal tumors. Human Pathol 1997; 27: 74-791. Bocker T, Bittinger A, Wieland W et al. In vitro and ex vivo expression of nucleolar proteins B23 and pl20 in benign and malignant epithelial lesions of the prostate. Modern Pathol 1995; 8: 226-31. Kallakury BVS, Sheehan CE, Rhee SJ et al. The prognostic significance of proliferation-associated nucleolar protein pi20 expression in prostate adenocarcinoma. Cancer 1999; 85' 156976. Kawai T, Suzuki M, Kono S et al. Proliferating cell nuclear antigen and K.i-67 in lung carcinoma. Correlation with DNA flow cytometric analysis. Cancer 1994; 74. 2468-75. Nishio M, KoshikawaT, Kuroishi Tet al. Prognostic significance of abnormal p53 accumulation in primary, resected non-smallcell lung cancers. J Clin Oncol 1996; 14. 497-502. Zhou X, Kemp BL, Khuri FR et al. Prognostic implication of
microsatelhte alteration profiles in early-stage non-small-cell lung cancer Clin Cancer Res 2000; 6: 559-65. 26. HashimotoT, Kobayashi Y. Ishikawn Yet al. Prognostic value of genetically diagnosed lymph node micrometastasis in non-smallcell lung carcinoma cases. Cancer Res 2000; 60: 6472-8. Received 26 March 2001: accepted 31 May 2001.
Correspondence to Y. Saijo, MD, PhD Department of Respiratory Oncology and Molecular Medicine Institute of Development, Aging, and Cancer Tohoku University 4-1 Seiryomachi Aobaku Sendai 980-77 Japan E-mail: [email protected]
Original article Expression of nucleolar protein pi20 predicts poor prognosis in patients with stage I lung adenocarcinoma Y. Saijo,1 G. Sato,1 K. Usui,1 M. Sato,2 M. Sagawa,2 T. Kondo, 2 Y. Minami 3 & T. Nukiwa1 ^Department of Respiratory Oncology and Molecular Medicine. 2 Department of Thoracic Surgery. Institute of Development. Aging, and Cancer. Tohoku University: ''Division of Epidemiology. Miyagi Cancer Center, Natori. Japan
Summary Background: P120 is a proliferation-associated nucleolar protein found in most human malignant tumors, but not in resting normal cells. In our previous studies, the expression of pl20 was statistically correlated with the proliferation capacity in human lung cancer cells and could be a prognostic marker for resected lung adenocarcinoma. Patients and methods: The expression levels of pl20 in tumors were assessed by immunohistochemistry in 59 patients with stage I lung adenocarcinoma who underwent radical resection. Using clinical follow-up data, the prognostic significance of pi20 calculated by labeling indices was evaluated using Cox's proportional hazard model. Results: A mean ± SD of the labeling index of pl20 was
Introduction Lung cancer is a major cause of cancer death worldwide. In Japan, deaths from lung cancer increase annually and accounted for more than 50,000 deaths in 1998 [1]. The overall five-year survival rates remain less than 20% [2]. Although the patients with stage I lung cancer show the best prognosis, about 30% relapse after surgical resection. Therefore, it would be important to identify stage I patients with an adverse prognosis and treat them with adjuvant chemotherapy. An additional predictor other than that of the TNM classification for identifying the patients with a high risk of recurrence in early stage non-small-cell lung cancer should be formulated to complement the current methods. PI 20, identified as a proliferation-associated nucleolar protein, is detected in a wide range of human tumors but not in quiescent cells or normal resting cells [3, 4]. Gene transfer experiments of pl20 into mammalian cells indicate a linkage between pl20 expression and the growth capacity of tumor cells [5, 6]. Nop2, a recently identified yeast homologue of human pl20, is required for the synthesis of the 60S ribosomal subunit and processing of the 27 S pre-rRNA to the mature 25 S rRNA [7-10]. Although no specific biochemical function of pl20 in eukaryotic cells has been assigned, the increased expression of p!20 in tumor cells suggests
35.3 ± 14.4%. No significant correlation was found between the expression levels of pi20 and clinicopathological factors. Using a cutoff value of 35% in the labeling index of pi20, patients with high expression of pl20 experienced early recurrence and shorter survival compared with those having low expression of pl20 (P = 0.04). Multivariate analysis revealed that pl20 served as an independent and strongest prognostic factor for resected lung adenocarcinoma (P = 0.033). Conclusion: This article provides the first evidence that the expression levels of pi20 in tumor tissues can be used as an independent and powerful prognostic marker for resected stage I lung adenocarcinoma. Key words: lung adenocarcinoma, nucleolar protein, pl20, prognosis, stage I
pl20 takes part in activated ribosomal biogenesis and accelerated cell growth. We have previously studied the significance of pl20 expression in human lung cancer in vitro and in vivo [11, 12]. The expression of pl20 was statistically correlated with the proliferation capacity in human lung cancer cells [11]. In 74 patients with lung adenocarcinoma, the expression levels of pi20 in tumor tissues evaluated by immunohistochemistry could predict the prognosis of patients with resected lung adenocarcinoma [12]. However, these patients comprised different stages (39 with pathologic stage I, 16 with pathologic stage II, and 19 with pathologic stage HI) and the median follow-up time was relatively short (38.5 months). Therefore, our purpose was to study the prognostic significance of pi 20 in lung adenocarcinoma at an early stage and with a longer follow-up in another set of patients with stage I lung adenocarcinoma. Our results demonstrate that the expression of pi20 is an independent prognostic marker in stage I lung adenocarcinoma. Patients and methods Patients and tissue samples We studied 59 patients with stage I lung adenocarcinoma who consecutively underwent surgical resection in the Hospital of the Institute of
1122 Development. Aging, and Cancer, Tohoku University, and affiliated hospitals from February 1991 to July 1996. All patients underwent surgery without pre-operative treatment. Informed consent was obtained from all patients before surgical resection. The patients consisted of 30 men and 29 women whose ages ranged from 45 to 76 years. According to the International System for Staging Lung Cancer [13], the cases comprised 42 patients with pathologic stage IA and 17 with pathologic stage IB. For the present analysis, the follow-up data were updated in July 2000 A minimum 48-month follow-up was obtained. During the follow-up. 15 patients died of lung cancer For routine histologic studies, tissues fixed with 10% formaldehyde were stained with hematoxylin and eosin.
lmmunohistochemical staining of pi 20
Fresh tumor samples were embedded in an ornithine carbamil transferase (OCT) compound (Tissue-Tek. Elkhart, Indiana), frozen in liquid nitrogen and stored at — 80 °C until use. Cryostat specimens were fixed with 2% formaldehyde and permeabilized with acetone. The samples were then incubated with mouse anti-human pl20 monoclonal antibody (pl20 MAb) (Oncogene Science, Cambridge, Massachusetts) overnight at 4°C [II]. lmmunohistochemical staining was performed using the streptavidin-biotin procedure with a Histofine SAB PO (M) kit (Nichirei, Tokyo). The tissues were finally counter-stained with Myer-hematoxylin solution Two examiners independently observed more than 500 cancer cells in more than 10 randomly selected fields and counted the pi 20 positive cancer cells. The labeling index of p!20 was defined as the percentage of cancer cells with pi 20 positive nucleoli among the total number of cancer cells counted. When a difference of the labeling index of pl20 was over 15% between two examiners, the index was re-evaluated. The results of the assessment for pl20 staining were blinded concerning the clinical data of the patients.
Statistical analysis All statistical analyses were carried out using the Abacus Concepts. Slat View (Abacus concepts. Inc.. Berkley, California). The chi-square test was used to examine the association between the labeling index of pl20 and several clinicopathological factors. The overall survival was calculated from the date of surgery, using as the end point death from any causes. Disease-free survival (DFS) was determined from the day of surgery, using as the end point the initial recurrence of the resected lung cancer assessed by clinical examinations. No patients died of other disease or were lost to follow-up. Overall survival and DFS curves were calculated using the Kaplan-Meier method and analyzed by the log rank test. The influence of each variable on survival was examined by the Cox's proportional hazards model in multivariate regression analyses. Statistical significance was defined as P < 0.05.
Results lmmunohistochemical staining of p 120 and clinicopathologicalfactors Nucleolar protein pl20 was clearly stained on the nucleoli of cancer cells by immunohistochemistry (Figure 1). In contrast, only nucleoli of alveolar macrophages were weakly stained in normal lung tissues. The expression levels of pl20 protein were quantified by counting the pl20-positive cancer cells in the tumor tissues. The labeling indices of pi20 among the tumor tissues varied from 9.7% to 64.7%, with a mean of 35.3 ± 14.4%. These indices were quite similar to those of our previous study
Figure I. lmmunohistochemical staining of nucleolar protein pl20 in resected lung adenocarcinoma. A frozen section of resected lung adenocarcinoma was stained with mouse monoclonal antibody pi20 as described in 'Patients and methods'. P120 was overexpressed in nucleoli of the cancer cells, but not in those of stromal (magnification x400).
[11]. No clinicopathological factors including gender, age, tumor size, and differentiation, were statistically correlated with the labeling index of pi 20 (Table 1). Expression ofpi 20 and prognosis in lung adenocarcinoma with stage I During the follow-up period (up to 60 months), 15 patients died of lung cancer. Post-operative overall survival and DFS curves in all cases according to the labeling indices of pl20 are shown in Figures 2a and 2b. When the cutoff value of the labeling index of pi 20 was chosen at 35%, patients with a high expression of pl20 showed a worse prognosis than those with low expression of pl20 (P - 0.04) (Figure 2a). Although median Table 1 Clinicopathological factors in 59 patients with stage I lung adenocarcinoma and labeling index of pi 20 in resected tumor tissues.
Gender Male Female Age < 65 5= 65 Tumor size Tl T2 Differentiation Well Moderate Poor Other
No. of patients
Labeling index ofpl20
29 30
36 9 ± 12.3" 33.9 ± 16.3
NS
28 31
36.4 ± 14.6 3 4 4 + 14.6
MS
42 17
36.2 ± 13.5 32 2 ± 16.7
34 12 9
35.4 ± 36.6 ± 31 5 ± 39 4 ±
4
Abbreviation' NS - not significant. ' Mean ± standard deviation.
15.4 12.7 13.6 86
P-value
NS
1123 Table 2. Mullivariate regression analyses of variables for predicting overall survival in patents with stage I lung adenocarcinoma.
(a) Lowpl20(n=28)
Variable
Hazard ratio
Unfavorable/favorable
f-value
Age Sex Tumor size Differentiation P120
2 607 0.158 2.958 0.675 10.417
>65/^65 male/female T2/T1 moderate, poor/well >35%/=S35%
0.183 0 111 0.111 0.412 0.033
Table 3 Multivariate regression analysis of variables for predicting disease free survival in patents with stage I lung adenocarcinoma. 10
20 30 40 Monthi after operation
50
(b)
Variable
Hazard ratio
Unfavorable/favorable
P-value
Age Sex Tumor size Differentiation P120
1.647 0.111 6.297 0.489 19.262
>65/<65 male/female T2/T1 moderate, poor/well > 35%/ s: 35%
0.498 0.074 0.024 0 484 0.012
Discussion
20 30 40 Monthi after operation
50
60
Figure 2. The Kaplan-Meier curves according to nucleolar protein pi 20 expression in patients with stage I lung adenocarcinoma Postoperative overall survival (A) and disease free survival (B) curves in 59 patients with stage-I lung adenocarcinoma were plotted based on a comparison between low (<35%) (28 cases) and high (3s 35%) (31 cases) labeling indices of pl20
survival times have not been reached in both the high pl20 expression group and the low pl20 expression group, the three- and five-year overall survival were 95% and 89% in the low pl20 expression group, but were only 77% and 65% in the high pl20 expression group. The patients with high pi20 expression tended to relapse earlier with high incidence resulting in shorter prognosis, but the p value of DFS was not significant (P = 0.07) (Figure 2b). The factors affecting the overall survival and DFS were analyzed by multivariate analysis using Cox's proportional hazards model. For overall survival, only pi20 was an independent prognostic factor (hazard ratio = 10.417, P = 0.033) among all variables (Table 2). Although both tumor size and pi20 were independent prognostic factors for DFS (Table 3), the hazard ratio of pl20 (19.26) was larger than that of the tumor status (6.297). Taken together, the expression of pi 20 was the strongest prognostic factor in the stage I lung adenocarcinoma.
The purpose of this study was to evaluate whether the expression level of pl20 in tumor tissues could predict the clinical outcome of patients with stage I lung adenocarcinoma. Our current study indicates that the expression of pi20 correlates with an early recurrence and poor prognosis after surgery. P120, a proliferation-associated nucleolar protein, is closely associated with the proliferative properties of mammalian cells. In human cancer cell lines, the expression of pl20 was documented to be associated with the S phase fraction of the cell cycle and doubling time [14]. The over-expression of pl20 in NIH3T3 cells by pl20 gene transfection enhanced cell proliferation [5]. In addition, down-regulation of pi20 expression by antisense pl20 gene caused growth inhibition of cancer cells [6, 15]. P120 was overexpressed more than 10-fold in tumor cells compared to normal resting cells. Although the specific transcriptional factor for pl20 has not been identified, the regulatory mechanisms of pi20 gene expression have been studied [16-18]. Mapping of the cisacting transcriptional control elements for the pl20 gene showed that, when the region containing the Alu sequences was deleted, transcription of the pl20 gene was reduced six-fold [16]. A recent report provides additional evidence that an RNA polymerase III transcriptional unit containing a homology to the highly repeated Alu sequence is located in the 5' flanking elements of the pl20 gene and that this positively regulates pi20 gene expression [18]. In situ expression of pi20 in malignant tissues has only been studied in a few human cancers including breast, prostate, colon, and lung cancer [11, 12, 19-23]. Freeman was the first investigator to study the expres-
1124 sion of pl20 in human cancer tissues and demonstrate a correlation between pl20 and the prognosis [19]. In 114 human breast cancer patients, they reported that 69% of the breast cancer tissues were positive for pi20, and that the immunopositivity of pl20 was correlated with patient survival, nodal status, estrogen receptor levels, and number of mitoses. Multivariate analysis showed that the worst prognosis was for patients who had tumor-positive nodes and tumor expressing pl20. This study concluded that pl20 was an independent and strongest prognostic marker in resected breast cancer. Ueki et al. reported increasing pl20 expression in accordance with the progression from mild dysplasia to severe dysplasia and carcinoma in human colorectal tumors [21]. They also demonstrated a significant correlation between pi 20 expression and another cell proliferation marker, Ki-67, but not with any known prognostic markers in colorectal carcinomas. Recently, the prognostic significance of pl20 in prostate carcinoma was reported [23]. One hundred thirty-two specimens of prostate carcinoma were stained with pl20 along with other proliferation markers. The expression of pl20 correlated with the post surgical recurrence in univariate analysis, but not in multivariate analysis. We have studied the expression of pl20 in human lung cancer using cancer cell lines and resected tumors. The expression of pi20 in six lung cancer cell lines was statistically correlated with their proliferation capacities [11]. In our previous immunohistochemical study of pi20 in resected lung adenocarcinoma, the expression level of pi20 was negatively correlated with the tumor doubling time (DT) determined by a retrospective radiographic calculation. Patients with a high labeling index for pi 20 had a high risk of early recurrence, resulting in a poor prognosis [12]. This study clearly shows that the expression of pl20 has prognostic value in stage I lung adenocarcinoma. The patients with a low labeling index of pl20 (<35%) had a statistically better survival rate than those with a high labeling index (^35%) (overall survival rate; 89% vs. 65%, P = 0.04). The multivariate analysis indicated that pi20 expression is the only prognostic indicator for overall survival and that pl20 expression and T status are significant indicators for disease-free survival in stage I lung adenocarcinoma. Many genetic markers have been analyzed as independent prognostic factors in stage I lung adenocarcinoma, such as K-ras, p53, microsattelite alterations, PCNA, Ki-67, etc. [23-26]. PCNA, ki-67 and pl20 are all proliferation markers. However, PCNA and ki-67 are localized in the nucleus whereas pl20 are localized in the nucleolus. Although prognostic value of pl20 in stage I lung adenocarcinoma was shown in this study, a further study in another set of patients should be evaluated to confirm the prognostic value of pl20. In addition, correlation of pl20 and other genetic markers should be studied in lung adenocarcinoma. Finally, we conclude that pl20 could predict the clinical outcome of the patients with stage I resected
lung adenocarcinoma by reflecting the tumor growth fractions.
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Correspondence to Y. Saijo, MD, PhD Department of Respiratory Oncology and Molecular Medicine Institute of Development, Aging, and Cancer Tohoku University 4-1 Seiryomachi Aobaku Sendai 980-77 Japan E-mail: [email protected]